This proposal will examine the novel hypothesis the genesis and function of breast cancer stem cells (CSCs) are governed by a feed forward loop that sustains activation of the Hippo transducer TAZ. The nexus of this loop is a splice variant of the ?6?1 integrin, ?6B?1. Two variants of the ?6 subunit exist (?6A and ?6B) that differ onl in their cytoplasmic domains. Alternative splicing of a common mRNA generates these variants. ?6A?1 integrin signaling prevents TAZ activation. The first aim will pursue the mechanism involved based on the findings that ?6A?1 interacts with the polarity/tumor suppressor protein Scribble and that Scribble associates with TAZ and prevents its nuclear localization. Thus, the first aim will test the hypothesis that the ?6A?1 integrin interacts with Scribble on the surfaceof mammary epithelial cells and contributes to its anchoring on the plasma membrane, an interaction mediated by the PDZ-binding domain present in the ?6A cytoplasmic domain. It is postulated that the ?6A integrin/Scribble complex sequesters TAZ preventing its nuclear localization and activation. The second aim will assess the hypothesis that one mechanism by which TAZ promotes the genesis of CSCs is to repress the mRNA splicing factor ESRP1 resulting in the genesis of ?6B?1 and loss of ?6A?1, and the formation of a LM511 matrix. Moreover, it is postulated that TAZ regulates VEGF transcription and induces VEGF signaling, and that VEGF signaling enables the BMI1-mediated repression of ESRP1. TAZ also promotes the formation of a LM511 matrix that functions as the ligand for ?6B?1. Consequently, a feed forward loop is established involving TAZ and LM511/?6B?1 signaling that sustains CSCs. The third specific aim will determine how ?6A?1 and ?6B?1 signaling differ in their ability to actiate TAZ focusing on the role of Jun-terminal kinase (JNK). The specific hypothesis to be addressed is that ?6A?1 signaling promotes JNK activation and JNK-mediated phosphorylation of TAZ on a novel serine (S90), which contributes to TAZ inactivation. LM511/?6B?1 signaling, in contrast, prevents JNK activation and JNK-mediated TAZ phosphorylation enabling TAZ activation. This hypothesis infers that JNK inhibition has a causal role in TAZ activation and the acquisition of stem cell properties, which will be evaluated. This proposal is rich in innovation and significance and the results to be obtained will have a major impact on our understanding of the biology of breast CSCs and reveal new mechanisms for therapeutic intervention.